Crude synaptic membrane of rat brain contains GABA(A) and GABA(B) receptors. We have examined whether GABA(A) or GABA(B) receptor sites or both are coupled to the recognition sites for benzodiazepines. The enchancement of Bmax of 3H-GABA binding at 37 degrees by diazepam (DIZ) was increased by EGTA in the binding assay. When the binding to GABA(A) and GABA(B) was differentiated by the presence of baclofen and THIP respectively, the binding to GABA(A) was enhanced by DIZ but the binding to GABA(B) was unaffected. Conversely the enhancement of 3H-flunitrazepam binding by GABA occurs only when GABA(A) receptor sites are free. Thus GABA(A) but not GABA(B) recognition sites are linked to the DIA recognition sites. In addition, we have found that in the crude synaptic membranes, various membrane-rigidifying compounds enhance the high affinity binding of 3H-GABA, whereas membrane-fluidizing factors decrease the binding. Moreover addition of free fatty acids or depolarization with K+ reduced the specific binding of 3H-GABA to intact NB2a cells. These results suggest that membrane-fluidity plays an important role in regulating GABA receptor function.